The end product of transglutaminase crosslinking: simultaneous quantitation of [Nepsilon-(gamma-glutamyl) lysine] and lysine by HPLC-MS3.
Transglutaminases catalyze the formation of Nepsilon-(gamma-glutamyl) isodipeptide crosslinks between proteins. These enzymes are thought to participate in a number of diseases, including neurological disease and cancer. A method associating liquid chromatography and multiple stage mass spectrometry has been developed for the simultaneous quantitation of [Nepsilon-(gamma-glutamyl) lysine] isodipeptide and lysine on an ion trap mass spectrometer. Highly specific detection has been achieved in MS3 mode. The method includes a derivatization step consisting of butylation of carboxylic groups and acetylation of amide groups, a liquid-liquid extraction, and a 19-min separation on a 100x2.1-mm Beta-basic C18 column with an acetonitrile gradient elution. 13C6-(15)N2 isotopes of the isodipeptide and the lysine serve as internal standards. The assay was linear in the range of 50 pmol/ml to 75 nmol/ml for the isodipeptide and the range of 10 nmol/ml to 3.5 micromol/ml for the lysine, with correlation coefficients greater than 0.99 for both ions. Intra- and inter-day coefficients of variation ranged from 3.5 to 15.9%. The method was successfully applied to human biological samples known to be crosslinked by transglutaminase such as cornified envelopes of epidermis, fibrin, and normal and Huntington disease brain.
Hoffner
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Laboratoire Régulation de la Transcription et Maladies Génétiques, UPR 2228 CNRS, Institut Interdisciplinaire des Sciences du Vivant des Saints-Pères, Université Paris Descartes, 75270 Paris Cedex 06, France. guylaine.hoffner@parisdescartes.fr
van der Rest
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G
Dansette
P M
PM
Djian
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P
D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004151:Dipeptides / Q000032:analysis*; D006801:Humans; D008239:Lysine / Q000032:analysis*; D013058:Mass Spectrometry / Q000379:methods*; D011503:Transglutaminases / Q000737:chemistry / Q000378:metabolism*
D003432:Cross-Linking Reagents; D004151:Dipeptides; C001299:epsilon-(gamma-glutamyl)-lysine; D011503:Transglutaminases; D008239:Lysine